HIV Viral Protein U (Vpu) is an essential, transmembrane viral protein with several functions in modulating the host cellular environment for optimal viral replication. First, Vpu plays a critical role in remodeling the cell surface by removing membrane-bound host proteins that inhibit viral replication, including CD4, BST-2/Tetherin, and MHC molecules. It does this by recruitment of the Cul1-?TrCP-Skp1-Rbx1 E3 ligase complex to the membrane, which subsequently ubiquitinates and removes target proteins from the cell surface by retrafficking and/or degradation. Second, Vpu is known to influence immune signaling, particularly through deregulation of NFKb. This occurs through both sequestration of ?TrCP, which would otherwise activate NFKb by degradation of its IKK inhibitor, and through down-regulation of BST-2, which is thought to activate NFKb through an interaction between its cytoplasmic domain and TRAF. Third and finally, Vpu is thought to act as a homo-oligomeric viroporin ion channel in the Golgi apparatus to alter membrane potential and potentially enhance virion release. All three functions rely on the coordination of multiple events at the cell membrane in conjunction with a series of characterized and yet unknown host protein complexes. An imperfect understanding of the host complexes involved and the inherent difficulties of working with membrane proteins in vitro has stifled our ability to understand how Vpu carries out each of these distinct processes. To better characterize the multifunctional nature of Vpu, we propose to employ an overall strategy that couples state-ofthe art proteomic discovery with structural/biophysical mechanistic interrogation and primary cell genetic validation. In Aim 1, we will employ global proteomic techniques including post-translational modification (PTM) profiling and Ascorbate Peroxidase-based proximity biotin labeling mass spectrometry (APEX-MS) to identify the host complexes and signaling pathways engaged by Vpu and select separation-of-function mutants (Core 1 and 5). In Aim 2, we will employ a combination of high-throughput mutagenesis and antibody-derived binding partner stabilization approaches to obtain cryo-EM and X-ray diffraction structures of monomeric and homo-oligomeric Vpu complexes at atomic resolution (Cores 3, 4, 6 and 7). Candidate host binding factors identified in Aim 1 will be tested for Vpu binding in vitro by Fluorescence Size Exclusion Chromatography (FSEC) and similarly used for structural interrogation. In Aim 3, we will employ primary cell CRISPR/Cas9 editing approaches to knock-out each of the candidate host factors identified in Aim 1 to test their impact on the replication of a series of vpu mutant viruses (Core 2). Protein-protein interactions and localization in the presence and absence of Vpu will be validated in vivo by confocal microscopy. Working with Core 5, our data will be collated for structure-function hypothesis generation that we will ultimately test in our primary cell genetic model. This project bridges expertise across the entire HARC collaborative and will generate structural and mechanistic insight into the multifunctional nature of HIV Vpu.